We are approaching this project by using homologous recombination in the mouse. A targeting vector containing an antibiotic resistance cassette flanked by inverted loxP sites was used to replace the entire epsilon-globin gene in mouse embryonic stem (ES) cells. We used recombinase mediated cassette exchange to replace the cassette with epsilon genes in which the promoter GATA-1, TATA or CACCC motifs had been destroyed or with a marked wild type epsilon gene as control. We also replaced the epsilon CACCC motif with the corresponding beta-globin promoter sequence with which the erythroid factor EKLF specifically interacts to activate adult beta-globin expression. In addition, we inserted a chromatin insulator, chicken 5 HS4, upstream of the epsilon gene separating the LCR from the structural genes. Differentiating ES cells express primarily epsilon-globin with a peak at about 12 days. Allelic discrimination and real-time qPCR were used to distinguish transcripts from the marked and unmutated epsilon-globin alleles. The GATA-1 and CACCC mutations, individually, were only modestly deleterious to epsilon-globin transcription, consistent with our studies of the human homologue. However, the beta-globin CACCC substitution was a very strong up-regulator of epsilon transcription, while, strikingly, insulator imposition upstream of epsilon-globin virtually eliminated its expression. Current studies using chromatin immunoprecipitation assays will address the effects of these mutations on chromatin structure of the locus and expression of the remaining genes. These studies are providing insights into the mechanisms underlying promoter specificity of LCR-globin gene interactions.